Research article Received: 13 September 2014,
Revised: 5 December 2014,
Accepted: 29 December 2014
Published online in Wiley Online Library: 24 February 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3433
Bioanalysis of acetylcarnitine in cerebrospinal fluid by HILIC–mass spectrometry Brian R. Holdera,b, Colleen A. McNaneya, David Luchettic, Eric Schaefferc and Dieter M. Drexlera* ABSTRACT: Acetyl-L-carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography– tandem mass spectrometry (HILIC-MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a highthroughput scale, a new and simple HILIC-MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via ‘dilute and shoot’ directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC-MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high-throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: LC-MS; acetylcarnitine; cerebrospinal fluid
Introduction
Biomed. Chromatogr. 2015; 29: 1375–1379
* Correspondence to: Dieter M. Drexler, Pharmaceutical Candidate Optimization, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Company, Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA. Email: [email protected] a
Pharmaceutical Candidate Optimization, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Company, Research and Development, 5 Research Parkway, Wallingford, CT, 06492, USA
b
Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, CT, 06340, USA
c
Exploratory Clinical and Translational Research, Bristol-Myers Squibb Company, Research and Development, 5 Research Parkway, Wallingford, CT, 06492, USA Abbreviations used: aCSF, artificial CSF; ALCAR, acetyl-L-carnitine; CNS, central nervous system; CSF, cerebrospinal fluid; HILIC, hydrophilic interaction liquid chromatography; rCSF, rat CSF; RT, room temperature.
Copyright © 2015 John Wiley & Sons, Ltd.
1375
The discovery and development of drugs for the treatment of neurological (central nervous system, CNS) diseases requires the measurement of specific biochemical changes and relevant endogenous metabolites (neurochemicals) in the brain, which may serve as biomarkers of disease or treatment effect. In preclinical research, these analytes can be monitored directly in brain tissue through either in vivo microdialysis techniques or ex vivo assays. However, these methods are not translatable to the clinic, and therefore developing assays that are suitable for measuring neurochemicals in the brain of animals and humans has become an area of increased focus. A relevant sample matrix for studying metabolites in neurological diseases is cerebrospinal fluid (CSF), which is in equilibrium with the brain’s interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Neurochemicals reflecting the pathophysiology of the brain are thus present in CSF and are amenable to in vivo analysis (Benitex et al., 2013). One such metabolite that has been linked to CNS diseases is acetyl-L-carnitine (ALCAR), an acetylated form of L-carnitine, which is involved in the transport of acetyl groups across the mitochondrial inner membrane. During strenuous exercise, L-carnitine is acetylated to ALCAR inside the mitochondria by carnitine O-acetyltransferase (Harris et al., 1987). The ALCAR is then transported outside the mitochondria, where it converts back to the two constituents. The L-carnitine is cycled back into the mitochondria with acyl groups to facilitate fatty acid utilization (Lysiak et al., 1988). ALCAR has been shown to be involved in energy metabolism in astrocytes and neurons, with the acetyl group being used in the synthesis of the neurotransmitters glutamate and GABA (Scafidi et al., 2010). Several studies have investigated whether administration of ALCAR can lead to beneficial effects in a variety of neurological conditions,
including dementia (Bonavita, 1986), depression (Tempesta et al., 1987) and neuropathic pain (Chiechio et al., 2002). The strongest evidence for a therapeutic effect is currently in neuropathic pain, where the analgesic effects of ALCAR have been shown to be mediated by the up-regulation of mGluR2 receptors in the lumbar spinal cord. ALCAR levels in serum have also been correlated with various disease or pathological states. A metabolomic analysis of plasma from schizophrenic patients demonstrated reduced levels of ALCAR in cases vs age-matched controls (He et al., 2012). Reduced serum levels of ALCAR have also been correlated with peripheral neuropathy in AIDS patients being treated with nucleoside analogs (Famularo et al., 1997), and in patients suffering from chronic fatigue syndrome (Kuratsune et al., 2002). Because of the relevance of ALCAR to a number of neurological conditions, we have developed an LC-MS assay employing
B. Holder et al. hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectroscopy (MS/MS) to measure the levels of this metabolite in the CSF of rodents, with the potential to be applied in the clinical setting.
Experimental Chemicals The analytical standard of ALCAR (CAS-5080-50-2) and the internal standard (IS), d3-ALCAR (CAS 362049-62-5) were from Sigma-Aldrich (St Louis, MO, USA). Artificial CSF or aCSF (perfusion fluid CNS) was from CMA Microdialysis Inc. (North Chelmsford, MA, USA). The CSF from rat (Sprague– Dawley), monkey (Cynomolgous), mouse (CD-1), dog (beagle) and human was obtained from Bioreclamation, LLC (Melville, NY, USA). A 1 mM stock solution of ALCAR was freshly prepared in HPLC-grade water, which was then diluted with aCSF to afford a 10,000 nM work solution. Further serial dilutions in aCSF yielded 13 calibration standards: 1500, 750, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 2, 1 and 0.5 nM. The d3-ALCAR was prepared at 1 mM in HPLC-grade water and diluted with water–acetonitrile (50:50 v/v) to prepare a 1000 nM IS solution. The ALCAR calibration standards and CSF samples were diluted with 1000 nM d3-ALCAR IS solution (50:50 v/v) in a 96-well Axygen PCR plate (Union City, CA) and sealed using a Axymat silicone cap mat (Union City, CA). The 96-well plate was shaken at 1000 rpm for 2 min to promote the denaturation and precipitation of endogenous proteins. It was then centrifuged at 3000 rpm at 4°C, affording pelletization of the proteins prior to injection of the supernatant onto the LC-MS/MS system.
temperature was set to 60°C. Chromatographic separation was achieved using mobile phases consisting of 15 mM ammonium acetate in water (aqueous mobile phase A) and acetonitrile (organic mobile phase B). The LC was performed at a flow rate of 600 μL/min and the sample injection volume was 10 μL. The initial gradient conditions were 10% A, held for 1 min, and a linear gradient was performed with mobile phase A increasing to 90% at 1.5 min and held for 2.5 min. The system was returned to initial conditions in 0.1 min and held for an additional 2.3 min for a total run time of 5 min. The mass spectrometer was operated in positive ion electrospray mode with source conditions as follows: ion spray voltage at 5000 V; ion source temperature at 500°C; declustering potential at 30 V; and collision energy at 15 eV for ALCAR. For the IS, the declustering potential and collision energy were 60 V and 25 eV, respectively. Selected reaction monitoring (MS/MS SRM) ion transitions were m/z 204.2 → 85.0 for ALCAR and m/z 207.0 → 85.0 for the IS (Scheme 1), as described previously (Longo et al., 1998; Magiera et al., 2013). Dwell times for the various transitions were set to 25 ms.
Data processing and ALCAR quantification AB Sciex Analyst software (version 1.6.1) was used to acquire and process all raw data. The calibration curve for ALCAR (average of three replicates) was generated by plotting the area ratios (analyte peak area/IS peak area) 2 against the known standard concentrations using a 1/x linear regression model. ALCAR levels in CSF were determined from the calibration curve.
Results and discussion
Assessment of the matrix effect
Current literature
The 10,000 nM ALCAR work solution was used to prepare 5000 and 2000 nM solutions in aCSF. A 5 μL aliquot from the 10,000, 5000 and 2000 nM solutions was used to separately spike 190 μL of blank aCSF for a final ALCAR concentration of 250, 125 and 50 nM, respectively. The percentage coefficient of variation (CV) was used to assess the inter-assay precision and the accuracy of ALCAR within each quality control (QC) standard for five replicate samples. The spiking procedure was repeated using rat CSF (rCSF) as a matrix. All spiked rCSF samples were analyzed for five replicates.
Previous studies have reported on the LC-MS quantification of ALCAR and d3-ALCAR in various biological fluids using elaborate sample pretreatments and various chromatographic stationary phases. Early on, Longo et al. (1998) incorporated deproteinization of human and animal plasma before analysis by an HILIC-MS/MS setup. The limit of quantitation (LOQ) for ALCAR and d3-ALCAR using this assay was established as 1000 nM. Later, Liu et al. (2008) performed protein precipitation and filtration of mouse plasma before analysis by an HILIC-MS/MS setup. The LOQ of d3-ALCAR in plasma was established as 4.8 nM. As part of a quantitative CSF metabolomic approach, Mandal et al. (2012) used a derivatization kit to quantify ALCAR in human CSF, employing direct flow injection-MS/MS analysis. For this derivatization assay, no information regarding the LOQ of ALCAR in CSF was provided. Filtration and deproteinization of urine and serum was performed by Isaguirre et al. (2013), before analysis by an HILIC-MS/MS setup. The LOQ of ALCAR in urine and serum was established as 3.2 and 4.3 nM, respectively. Solid-phase extraction of urine was performed by Magiera et al. (2013) prior to analysis by a reverse-phase (RP) LC-MS/MS setup. The LOQ of ALCAR in urine was established as 0.5 nM.
Stability of ALCAR The stability of ALCAR was assessed at room temperature (RT) and during freeze–thawing of rCSF samples. An aliquot of rCSF was kept at RT for 4 h, while another aliquot of rCSF was subjected to three successive freeze–thaw cycles. This experiment was performed for four replicates. To supplement the ALCAR stability HILIC-MS/MS findings, the ALCAR 1 levels in a 1 mM ALCAR frozen standard was monitored using H NMR over a duration of 3 months and also after three freeze–thaw cycles.
ALCAR in animal CSF Bioreclamation animal CSF from various animal models – rat, monkey, dog, mouse and human – were diluted with 1000 nM d3-ALCAR IS (50:50 v/v) for three biological replicates and subjected to HILIC-MS/MS analysis.
HILIC-MS/MS conditions
1376
A CTC HTS PAL autosampler (Leap Technologies, Carrboro, NC, USA), a 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA, USA) and an AB Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) were components of the LC-MS system. The LC was performed using a Waters Cortecs UHPLC HILIC column 2.1 × 100 mm, 1.6 μm (Waters, Milford, MA, USA). The column
wileyonlinelibrary.com/journal/bmc
Scheme 1. Proposed MS/MS fragmentation pattern of acetyl-L-carnitine (ALCAR).
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1375–1379
LC-MS bioanalysis of acetylcarnitine Method development With a potential application for clinical studies, there was a need to develop a robust LC-MS assay for ALCAR in CSF with minimal sample treatment amenable to large sample sets. For this study, ‘dilute and shoot’ sample preparation was employed prior to HILIC-MS/MS analysis. As an alternative to traditional RP stationary phases, HILIC columns have shown improved orthogonal selectivity for polar analytes. The advancement in HILIC technology with 1.6 μm solid-core particles increases stereoselectivity, thereby resulting in greater efficiency of chromatographic retention and separation and, in this case, an improved detection of ALCAR. For method development, aCSF was used as a surrogate matrix with ALCAR (including IS d3-ALCAR) being spiked at various concentrations. As shown in Fig. 1(A), blank aCSF showed a negligible MS response. The lower limit of quantitation (LLOQ) was determined to be at 0.5 nM ALCAR with a signal-to-noise ratio of ~ 5 at 2.2 min RT (Fig. 1B). The IS is also displayed (Fig. 1C).
Linearity ALCAR standards in aCSF were quantified using a 1/x2 weighted linear regression. The area ratios (analyte peak area/IS peak area) of three experiments were averaged to yield a calibration curve (Fig. 2) with a linear dynamic range of 0.56 (LLOQ) to 1500 nM and an r2 value of 0.99, validating the linear fit. The ALCAR calibration curve was used to determine the levels of ALCAR in all CSF samples.
experiments for each QC standard. All QC standards were found to be closely similar to the target concentrations with 96–100% accuracies and low CVs of
Revised: 5 December 2014,
Accepted: 29 December 2014
Published online in Wiley Online Library: 24 February 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3433
Bioanalysis of acetylcarnitine in cerebrospinal fluid by HILIC–mass spectrometry Brian R. Holdera,b, Colleen A. McNaneya, David Luchettic, Eric Schaefferc and Dieter M. Drexlera* ABSTRACT: Acetyl-L-carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography– tandem mass spectrometry (HILIC-MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a highthroughput scale, a new and simple HILIC-MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via ‘dilute and shoot’ directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC-MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high-throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: LC-MS; acetylcarnitine; cerebrospinal fluid
Introduction
Biomed. Chromatogr. 2015; 29: 1375–1379
* Correspondence to: Dieter M. Drexler, Pharmaceutical Candidate Optimization, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Company, Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA. Email: [email protected] a
Pharmaceutical Candidate Optimization, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Company, Research and Development, 5 Research Parkway, Wallingford, CT, 06492, USA
b
Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, CT, 06340, USA
c
Exploratory Clinical and Translational Research, Bristol-Myers Squibb Company, Research and Development, 5 Research Parkway, Wallingford, CT, 06492, USA Abbreviations used: aCSF, artificial CSF; ALCAR, acetyl-L-carnitine; CNS, central nervous system; CSF, cerebrospinal fluid; HILIC, hydrophilic interaction liquid chromatography; rCSF, rat CSF; RT, room temperature.
Copyright © 2015 John Wiley & Sons, Ltd.
1375
The discovery and development of drugs for the treatment of neurological (central nervous system, CNS) diseases requires the measurement of specific biochemical changes and relevant endogenous metabolites (neurochemicals) in the brain, which may serve as biomarkers of disease or treatment effect. In preclinical research, these analytes can be monitored directly in brain tissue through either in vivo microdialysis techniques or ex vivo assays. However, these methods are not translatable to the clinic, and therefore developing assays that are suitable for measuring neurochemicals in the brain of animals and humans has become an area of increased focus. A relevant sample matrix for studying metabolites in neurological diseases is cerebrospinal fluid (CSF), which is in equilibrium with the brain’s interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Neurochemicals reflecting the pathophysiology of the brain are thus present in CSF and are amenable to in vivo analysis (Benitex et al., 2013). One such metabolite that has been linked to CNS diseases is acetyl-L-carnitine (ALCAR), an acetylated form of L-carnitine, which is involved in the transport of acetyl groups across the mitochondrial inner membrane. During strenuous exercise, L-carnitine is acetylated to ALCAR inside the mitochondria by carnitine O-acetyltransferase (Harris et al., 1987). The ALCAR is then transported outside the mitochondria, where it converts back to the two constituents. The L-carnitine is cycled back into the mitochondria with acyl groups to facilitate fatty acid utilization (Lysiak et al., 1988). ALCAR has been shown to be involved in energy metabolism in astrocytes and neurons, with the acetyl group being used in the synthesis of the neurotransmitters glutamate and GABA (Scafidi et al., 2010). Several studies have investigated whether administration of ALCAR can lead to beneficial effects in a variety of neurological conditions,
including dementia (Bonavita, 1986), depression (Tempesta et al., 1987) and neuropathic pain (Chiechio et al., 2002). The strongest evidence for a therapeutic effect is currently in neuropathic pain, where the analgesic effects of ALCAR have been shown to be mediated by the up-regulation of mGluR2 receptors in the lumbar spinal cord. ALCAR levels in serum have also been correlated with various disease or pathological states. A metabolomic analysis of plasma from schizophrenic patients demonstrated reduced levels of ALCAR in cases vs age-matched controls (He et al., 2012). Reduced serum levels of ALCAR have also been correlated with peripheral neuropathy in AIDS patients being treated with nucleoside analogs (Famularo et al., 1997), and in patients suffering from chronic fatigue syndrome (Kuratsune et al., 2002). Because of the relevance of ALCAR to a number of neurological conditions, we have developed an LC-MS assay employing
B. Holder et al. hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectroscopy (MS/MS) to measure the levels of this metabolite in the CSF of rodents, with the potential to be applied in the clinical setting.
Experimental Chemicals The analytical standard of ALCAR (CAS-5080-50-2) and the internal standard (IS), d3-ALCAR (CAS 362049-62-5) were from Sigma-Aldrich (St Louis, MO, USA). Artificial CSF or aCSF (perfusion fluid CNS) was from CMA Microdialysis Inc. (North Chelmsford, MA, USA). The CSF from rat (Sprague– Dawley), monkey (Cynomolgous), mouse (CD-1), dog (beagle) and human was obtained from Bioreclamation, LLC (Melville, NY, USA). A 1 mM stock solution of ALCAR was freshly prepared in HPLC-grade water, which was then diluted with aCSF to afford a 10,000 nM work solution. Further serial dilutions in aCSF yielded 13 calibration standards: 1500, 750, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 2, 1 and 0.5 nM. The d3-ALCAR was prepared at 1 mM in HPLC-grade water and diluted with water–acetonitrile (50:50 v/v) to prepare a 1000 nM IS solution. The ALCAR calibration standards and CSF samples were diluted with 1000 nM d3-ALCAR IS solution (50:50 v/v) in a 96-well Axygen PCR plate (Union City, CA) and sealed using a Axymat silicone cap mat (Union City, CA). The 96-well plate was shaken at 1000 rpm for 2 min to promote the denaturation and precipitation of endogenous proteins. It was then centrifuged at 3000 rpm at 4°C, affording pelletization of the proteins prior to injection of the supernatant onto the LC-MS/MS system.
temperature was set to 60°C. Chromatographic separation was achieved using mobile phases consisting of 15 mM ammonium acetate in water (aqueous mobile phase A) and acetonitrile (organic mobile phase B). The LC was performed at a flow rate of 600 μL/min and the sample injection volume was 10 μL. The initial gradient conditions were 10% A, held for 1 min, and a linear gradient was performed with mobile phase A increasing to 90% at 1.5 min and held for 2.5 min. The system was returned to initial conditions in 0.1 min and held for an additional 2.3 min for a total run time of 5 min. The mass spectrometer was operated in positive ion electrospray mode with source conditions as follows: ion spray voltage at 5000 V; ion source temperature at 500°C; declustering potential at 30 V; and collision energy at 15 eV for ALCAR. For the IS, the declustering potential and collision energy were 60 V and 25 eV, respectively. Selected reaction monitoring (MS/MS SRM) ion transitions were m/z 204.2 → 85.0 for ALCAR and m/z 207.0 → 85.0 for the IS (Scheme 1), as described previously (Longo et al., 1998; Magiera et al., 2013). Dwell times for the various transitions were set to 25 ms.
Data processing and ALCAR quantification AB Sciex Analyst software (version 1.6.1) was used to acquire and process all raw data. The calibration curve for ALCAR (average of three replicates) was generated by plotting the area ratios (analyte peak area/IS peak area) 2 against the known standard concentrations using a 1/x linear regression model. ALCAR levels in CSF were determined from the calibration curve.
Results and discussion
Assessment of the matrix effect
Current literature
The 10,000 nM ALCAR work solution was used to prepare 5000 and 2000 nM solutions in aCSF. A 5 μL aliquot from the 10,000, 5000 and 2000 nM solutions was used to separately spike 190 μL of blank aCSF for a final ALCAR concentration of 250, 125 and 50 nM, respectively. The percentage coefficient of variation (CV) was used to assess the inter-assay precision and the accuracy of ALCAR within each quality control (QC) standard for five replicate samples. The spiking procedure was repeated using rat CSF (rCSF) as a matrix. All spiked rCSF samples were analyzed for five replicates.
Previous studies have reported on the LC-MS quantification of ALCAR and d3-ALCAR in various biological fluids using elaborate sample pretreatments and various chromatographic stationary phases. Early on, Longo et al. (1998) incorporated deproteinization of human and animal plasma before analysis by an HILIC-MS/MS setup. The limit of quantitation (LOQ) for ALCAR and d3-ALCAR using this assay was established as 1000 nM. Later, Liu et al. (2008) performed protein precipitation and filtration of mouse plasma before analysis by an HILIC-MS/MS setup. The LOQ of d3-ALCAR in plasma was established as 4.8 nM. As part of a quantitative CSF metabolomic approach, Mandal et al. (2012) used a derivatization kit to quantify ALCAR in human CSF, employing direct flow injection-MS/MS analysis. For this derivatization assay, no information regarding the LOQ of ALCAR in CSF was provided. Filtration and deproteinization of urine and serum was performed by Isaguirre et al. (2013), before analysis by an HILIC-MS/MS setup. The LOQ of ALCAR in urine and serum was established as 3.2 and 4.3 nM, respectively. Solid-phase extraction of urine was performed by Magiera et al. (2013) prior to analysis by a reverse-phase (RP) LC-MS/MS setup. The LOQ of ALCAR in urine was established as 0.5 nM.
Stability of ALCAR The stability of ALCAR was assessed at room temperature (RT) and during freeze–thawing of rCSF samples. An aliquot of rCSF was kept at RT for 4 h, while another aliquot of rCSF was subjected to three successive freeze–thaw cycles. This experiment was performed for four replicates. To supplement the ALCAR stability HILIC-MS/MS findings, the ALCAR 1 levels in a 1 mM ALCAR frozen standard was monitored using H NMR over a duration of 3 months and also after three freeze–thaw cycles.
ALCAR in animal CSF Bioreclamation animal CSF from various animal models – rat, monkey, dog, mouse and human – were diluted with 1000 nM d3-ALCAR IS (50:50 v/v) for three biological replicates and subjected to HILIC-MS/MS analysis.
HILIC-MS/MS conditions
1376
A CTC HTS PAL autosampler (Leap Technologies, Carrboro, NC, USA), a 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA, USA) and an AB Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) were components of the LC-MS system. The LC was performed using a Waters Cortecs UHPLC HILIC column 2.1 × 100 mm, 1.6 μm (Waters, Milford, MA, USA). The column
wileyonlinelibrary.com/journal/bmc
Scheme 1. Proposed MS/MS fragmentation pattern of acetyl-L-carnitine (ALCAR).
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1375–1379
LC-MS bioanalysis of acetylcarnitine Method development With a potential application for clinical studies, there was a need to develop a robust LC-MS assay for ALCAR in CSF with minimal sample treatment amenable to large sample sets. For this study, ‘dilute and shoot’ sample preparation was employed prior to HILIC-MS/MS analysis. As an alternative to traditional RP stationary phases, HILIC columns have shown improved orthogonal selectivity for polar analytes. The advancement in HILIC technology with 1.6 μm solid-core particles increases stereoselectivity, thereby resulting in greater efficiency of chromatographic retention and separation and, in this case, an improved detection of ALCAR. For method development, aCSF was used as a surrogate matrix with ALCAR (including IS d3-ALCAR) being spiked at various concentrations. As shown in Fig. 1(A), blank aCSF showed a negligible MS response. The lower limit of quantitation (LLOQ) was determined to be at 0.5 nM ALCAR with a signal-to-noise ratio of ~ 5 at 2.2 min RT (Fig. 1B). The IS is also displayed (Fig. 1C).
Linearity ALCAR standards in aCSF were quantified using a 1/x2 weighted linear regression. The area ratios (analyte peak area/IS peak area) of three experiments were averaged to yield a calibration curve (Fig. 2) with a linear dynamic range of 0.56 (LLOQ) to 1500 nM and an r2 value of 0.99, validating the linear fit. The ALCAR calibration curve was used to determine the levels of ALCAR in all CSF samples.
experiments for each QC standard. All QC standards were found to be closely similar to the target concentrations with 96–100% accuracies and low CVs of
